The long-term objective of this project is to provide an as complete as possible kinetic and thermodynamic description of the sodium-potassium pump, or (Na+K)ATPase. The sodium-potassium pump is present in the membrane of nearly all animal cells, where it is responsible for maintaining the transmembrane Na and K gradients which underlie nerve and muscle excitation, volume and pH regulation, fluid secretion, sensory transduction, secondary transport of nutrients and other ions, etc. The electrogenic nature of the pump (it expels more sodium than it imports potassium) plays a central role in sensory adaptation, in pacemaker generation, and in synaptic modulation. The action of digitalis drugs on the heart is almost certainly through the sodium pump. The sodium pump in man consumes 20-50% of all ATP made in the body. The cell chosen for this work is a giant nerve fiber of the squid which, because of its very large size, allows experimental access and control not available with other cells. From the brain of the squid, the (Na+K)ATPase enzyme will be extracted, and its enzymic properties compared with the transport properties of the in situ axon pump. The specific aims for this proposal are to establish, wherever possible, kinetic correlations between "biophysical" events mediated by the pump, such as the various transport modes known as Na:K exchange, K:Na exchange or reverse pumping, Na:Na exchange, K:K exchange, and "uncoupled" Na efflux, with "biochemical" events catalyzed by the same enzyme, such as ATP hydrolysis, ADP:ATP exchange, ATP-enhanced de-occlusion of "occluded K", enzyme phosphorylation and dephosphorylation. Transport across the axon membrane will be studied using the internal dialysis technique, whereby the cell interior and exterior are put under complete experimental control, and isotope fluxes in either direction can be measured. Membrane potential will be controlled by means of a very stable (plus and minus 40 MuV) voltage-clamp circuit. The electric current generated by the pump will be measured as the cardiotonic steroid induced change in holding current. Systematic variations of one variable at a time (external or internal Na, K, Mg or metabolite concentration, pH, or membrane potential) will yield extensive sets of kinetic data curves which must then be analyzed by least squares methods within the framework of the known biochemistry of the (Na+K)ATPase.